The invention concerns a method for intracellular manipulation of at least one deposited biological cell situated in a cultural medium adhering to a support area, wherein an opening is created in the membrane of the cell, and the cell interior is manipulated through this opening. The invention furthermore relates to an apparatus for manipulation of the cell interior of at least one biological cell having a cell membrane situated in a culture medium. The apparatus has an object carrier, which has at least one support area on which the cell is adherently depositable, a poration tool for opening the cell membrane, and at least one entry channel situated in the area of the poration tool for manipulation of the cell interior.
An apparatus of the type mentioned at the beginning is already known from Alberts, B. et al., Molecular Biology of the Cell, Third Printing, VCH Verlag (1995), page 212 ff, which has a hollow needle connected with a suction device and made of an electrical insulating material having an interior cavity which has an opening on the free end of the hollow needle. For opening the cell membrane, the opening situated at the free end of the hollow needle is set upon the exterior of the cell membrane in order then to create an underpressure in the interior cavity of the hollow needle by means of the suction device. Through this underpressure, a cell membrane piece situated in front of the opening of the hollow needle is torn out of the membrane formation. After introducing the opening into the cell membrane the hollow needle, engaging the edge of the cell membrane surrounding the opening, electrically insulates the cell fluid contained in the interior of the cell against the culture medium. The interior of the cell is manipulated through the opening introduced into the cell membrane. For example, the cell nucleus can be removed by sucking cell fluid into the hollow needle from the cell, and subsequently another cell nucleus can be installed through the opening into the cell interior.
The previously known process and the apparatus for implementing the process have the disadvantage that a micromanipulator is necessary for positioning the hollow needle on the cell. This results in a comparatively more complicated and expensive device. Moreover, the accessibility of the cells situated on the object carrier by the micromanipulator is greatly reduced. The process and the apparatus are, for this reason, only suited for an intracellular manipulation of individual, or at best for a simultaneous manipulation of a small number of cells situated on the object carrier. At the same time, a costly, manual positioning of the hollow needle on the cell is necessary.
Arranging a cell floating in a culture medium between two large surface area electrodes, respectively spaced at a distance from the cell, and applying an electric voltage to these electrodes is also already known from Alberts, B. et al., Molecular Biology of the Cell, Third printing, VCH Verlag (1995), page 213. Moreover, the membrane of the cell is simultaneously opened at several places by electroporation so that the genetic material situated in the culture medium to be introduced into the cell can be diffused into the cell interior through the openings of the cell. In this connection, however, it is disadvantageous that such an introduction of genetic material is subject to statistical fluctuations and is influenced by various parameters such as the concentration of genetic material in the area of the cell, the size of the genetic material to be introduced, and the size of the openings created in the cell membrane. The previously known process consequently does allow any selective manipulation of the cell interior.
There thus exists an object of creating a process and an apparatus of the type mentioned at the beginning, which make a simple manipulation of the interior of a cell possible. In particular, an expensive manual positioning of a hollow needle on the cell to be processed is to be avoided. This objective is accomplished according to the process of the invention in that the opening is introduced into the cell membrane within the support area of the cell and at a distance from its support edge.
In this manner, it is possible to arrange a poration agent for creating the cell membrane opening or a poration tool in the support area of the cell on the object carrier, so that when the cell is deposited on the support area, it is at the same time positioned on the poration agent or the poration tool. In this manner, an expensive manual positioning of a poration tool can be omitted. Since the opening is introduced into the cell membrane within the support area of the cell and at a distance from the edge of the support area, the membrane area of the cell surrounding the opening and adhering to the support area for the cell seals the opening against the culture medium. The cell fluid situated in the interior of the cell is thereby electrically insulated against the culture medium as extensively as possible, so that a potential equilization between the cell potential in the interior of the cell and that of the culture medium is prevented. Through the opening created into the cell membrane, the cell interior can be manipulated.
With an especially advantageous embodiment of the invention, the opening is introduced into the cell membrane by means of electroporation. While conducting the process, the electroporation electrode can, for example, be arranged in the support area on which the adhering cell lies . To introduce the opening into the cell membrane, only an electrical voltage then needs to be applied between the electroporation electrode and the culture medium. This brings about a flow of electrical current which opens the cell membrane.
With another embodiment of the invention, at least one mechanical impulse is exerted to introduce the opening into the cell membrane on a partial area of the cell membrane. In this connection, this partial area is released from the membrane formation. Optionally, an impulse succession can also be applied with several individual impulses.
It is especially advantageous if the opening is introduced into the cell membrane by means of sound waves, especially ultrasound and/or hypersound waves. Here, it is even possible for the sound waves to be focused on the area of the cell membrane to be opened and/or for several sound waves to be superimposed, such that their oscillations in the area of the cell membrane are overlaid into an oscillation with increased amplitude. The cell membrane can thereby be opened without contact.
A contact-free opening of the cell membrane can, however, also take place in that a partial area of the cell membrane is irradiated with energy-rich radiation, especially with laser radiation. In this connection, the wave length of the radiation is preferably selected such that the cell membrane absorbs the radiation well. Expediently, the radiation is launched into the cell at its support area. Optionally, however, a laser beam can also be launched into the cell outside the support area, in that first of all a small launch opening is introduced in a membrane area situated there. The laser beam is subsequently projected through the opening and through the interior of the cell to a membrane area situated in the support area of the cell, in order to cut out a partial area of the membrane from the membrane formation by swivelling the laser beam around the launch opening.
With another embodiment of the process, the opening is introduced into the cell membrane by the action of a chemical poration substance. Performing or Triton(copyright), for example, can be used as poration agents.
It is especially advantageous if an electrical and/or chemical and/or a radiation-activatable chemical substance is used, and if this substance for introducing the opening into the cell membrane is activated by the action of radiation, a chemical and/or an electrical field. The substance is thus activated by supplying energy. In this manner, free radicals can be generated, for example, which destroy the partial area of the cell membrane to be opened. In the inactive state, the substance behaves largely neutrally toward the cell so that it practically does not influence placing the cell on the support area. A chemical substance which is activated by administering a further substance can also be used.
Another embodiment of the process provides that a partial area of the cell membrane to be opened is released from the membrane formation with an underpressure and/or an overpressure. For this purpose, for example, a small opening can be provided inside the support area on an object carrier having the support area. Suction is applied through the opening to the cell so strongly that the membrane area situated in front of the opening is torn out of the membrane formation. For opening the cell membrane, an overpressure impulse can also be exerted through the opening on a membrane area of the cell. It is advantageous for the cell to be fixed on the support area by the force of suction. In this manner the adhesion of the cell to the support area of the object carrier can be improved. The force of suction is here so proportioned that the cell membrane is not mechanically damaged by the force of the suction.
It is advantageous if after introducing the opening into the cell membrane at least one substance and/or one cell component is removed from the cell interior and/or placed in the cell interior though the opening. Thus, for example, a medication, a protein and/or another biologically active substance can be brought through the opening into the interior of the cell in order to test its reaction. However, a gene or gene fragment can also be removed from the cell or introduced into it. Even the cell nucleus can be removed from the cell through the opening and replaced by another.
With regard to the apparatus of the invention, the accomplishment of the object mentioned above consists in that the poration tool is arranged within the support area. The cell can thereby be deposited on the poration tool situated in the support area. Advantageously, an expensive manual positioning of the poration tool on the cell is thereby avoided. Furthermore, no auxiliary devices, for example micromanipulators, are needed. It is thus possible to manipulate intracellularly several cells arranged tightly adjacent to one another in the support area at the same time. After introducing the opening into the cell membrane, the edge of the cell membrane surrounding the opening remains in contact with the support area of the object carrier and seals the opening of the cell membrane against the culture medium
An especially advantageous embodiment of the invention provides that the poration tool is bounded by at least one electrical insulator within the support area. By means of the electrical insulator, the cell fluid situated in the interior of the cell is then well insulated against the culture medium so that the interior of the living cell can be manipulated over a longer period of time without the cell dying off. The insulation resistance is dependent upon the cell type and is preferably greater than 10 megaohm. A potential equalization between the culture medium and the cell fluid is thereby prevented to the greatest extent. Optionally, the object carrier can consist wholly of an insulating material. The insulator can also, however, be arranged in the interior of the object carrier at a distance from the surface of the support area.
It is advantageous if the poration tool substantially concentrically surrounds the opening of the passage channel situated in the support area of the object carrier. The cell interior can then be even better manipulated through the opening without the edge area of the cell membrane surrounding the opening being damaged by the manipulation.
It is especially advantageous if the poration tool is an electroporation electrode, to which a reference electrode capable of being brought into contact with the culture medium is allocated, and if the electroporation electrode and the reference electrode are connectable for electroporation of the cell membrane with a source of electrical voltage. The electroporation electrode is arranged within the support area of the object carrier so that a cell adherently deposited on the insulator can likewise be deposited on the electrode, or at least can approach this up to the radius of action of an electrical field emanating from the electrode. When electroporation voltage is applied to the electrode, an electric current flows which introduces an opening into the cell membrane. At the same time, the edge of the cell membrane surrounding the opening remains in contact with the support area and seals off the opening against the culture medium. After creating the opening into the cell membrane, the electrode is separated from the electroporation voltage source, so that the interior of the cell can then be altered through the opening.
With one advantageous embodiment of the apparatus, the poration tool for opening the cell membrane is movable by means of an actuator, especially a piezo element, transversely to the surface of the support area relative to the object carrier. With this device, the opening is thus mechanically introduced into the cell membrane. The poration tool can thus be alternatively moved toward and away from the opening on the cell membrane. For this purpose, the actuator is connected with an activation facility for generating a mechanical vibration, especially an ultrasound or hypersound vibration. The poration tool can be mobile in a direction running at right angles or obliquely to the surface of the support area relative to the object carrier.
It is advantageous if the poration tool has at least one sharp tip or edge, preferably protruding relative to the surface plane of the support area. The poration tool can then optionally lie on the cell membrane, so that this can be mechanically opened even better. If the poration tool is an electrode for electroporation of the cell membrane, there results an especially high electric field strength when a poration voltage is applied to the electrode, which facilitates opening the cell membrane.
It is especially advantageous if a laser beam is provided as poration tool, and if the radiation path of the laser beam is passed through the entry channel to the opening situated in the support area. With this device, a partial area of the cell membrane can be irradiated for a short time with energy-rich optical radiation, wherein the latter is so strongly heated that the cell membrane opens. Optionally, beam guidance means can be arranged inside or outside the entry channel.
It is especially advantageous if a laser diode is incorporated into the object carrier for generating the laser beam. In this manner, the laser diode can even be arranged directly behind the opening of the entry channel so that the laser radiation can be launched directly and consequently largely free of loss into the cell membrane of the cell deposited on the support area.
It is provided with an advantageous embodiment of the invention that the poration tool for opening the cell membrane has a chemical poration substance in the support area of the object carrier and/or that a chemical poration substance can be fed in through the entry channel to its opening. The opening can thus also be introduced chemically into the cell membrane, whereby Performing or Triton(copyright) can be used as poration agents. Optionally, the entry channel can be connected or connectable with a deposit containing the poration substance, from which the poration substance is feedable to the cell membrane.
With another embodiment, the device has at least one pump which, for opening the cell membrane by contacting with underpressure or overpressure, is connected with the entry channel and/or the entry channel is connectable with an underpressure or overpressure reservoir through a valve or similar shut-off element. The opening can be introduced into the cell membrane through underpressure or overpressure, wherein the underpressure or overpressure is shut off following opening the cell membrane. In this manner, sucking off cell fluid from the interior of the cell during opening the cell membrane through underpressure is avoided to the greatest extent. Correspondingly, when opening the cell membrane using overpressure, any medium situated in the entry channel (which is preferably a fluid) can be prevented from reaching into the interior cell. For turning off the underpressure or overpressure, a pressure change occurring in the entry channel when opening the cell membrane can be determined. Advantageously, the entry channel can be also used before depositing the cell, in order to suck off culture medium from the support area, so that a current arises in the culture medium which leads the cells situated therein to the opening of the entry channel arranged in the area of the poration tool.
An advantageous embodiment provides that the poration tool is arranged on a projection protruding in relation to the surface plane of the support area. In this manner, there results a good electrical and/or mechanical contact between the poration tool and the cell membrane.
Expediently, it is provided that the cross section of the projection tapers at the furthest projecting point. The cell then adheres especially well on the support area of the object carrier in the area of the projection. Moreover, in terms of production engineering the insulator can be better applied during production of the object carrier as a coating to the tapering area of the projection.
With an advantageous refinement of the invention, it is provided that the object carrier has a contour in the support area, which has at least one profile depression running around the poration tool and/or one profile projection running around the poration tool. In this manner, a better sealing of the cell fluid against the culture medium by the cell membrane adhering to the insulator is obtained.
It is advantageous if the profile depression and/or the profile projection is interrupted by at least one gap in the projection direction. The cell can then adhere better in the area of the contouring to the surface of the object carrier. The profile projection or the profile depression can, for example, have a honeycomb structure, or a structure in the manner of a checker or chess board pattern.
It is especially advantageous if the profile depression and/or profile projection is constructed as a ring-shape, and if preferably several such annular profile depressions and/or profile projections are arranged substantially concentrically to the poration tool. Consequently, several profile depressions and projections are connected or inserted one after the other radially to the poration tool, so that the cell fluid is even better sealed off against the culture medium.
A preferred embodiment of the invention provides that the electrical insulator is an insulation layer arranged on the surface of the contour. Advantageously, with the insulation layer contoured in this manner, the path for a creeping current flowing on the surface of the insulator from the cell fluid to the culture medium is enlarged, so that the cell fluid situated in the interior of the cell is even better insulated against the culture medium after opening the cell membrane.
Another embodiment provides that the profile projection(s) is (are) installed on the surface of the electrical insulator. The object carrier is then easier to manufacture in terms of assembly.
It is especially advantageous if a cell coating having at least one cell adhesion protein and/or a hydrophilic coating and/or immediately adjacent to the poration tool a hydrophobic coating is arranged in the support area of the object carrier. The cell membrane then adheres better to the object carrier. The cell adhesion coating can include, for example, laminin, fibronectin or poly-L-lysine. Optionally, a hydrophobic coating with binding sites for hydrophobic lipids found in the cell membrane can also be arranged on the edge of the support area bordering on the electrode.
It is advantageous if, as a mechanical guide for the cells, boundary walls are arranged on both sides of the poration tool, which preferably delimit a groove-like guide channel. Moreover, the poration tool is preferably arranged in the middle between the boundary walls at the bottom of the groove of the guide channel, so that cells situated in the guide channel can essentially move only in the direction of extension of the guide channel and then necessarily come into contact with the poration tool.
It is advantageous if, for creating an electrical field leading the cell to the poration tool, at least one supplemental electrode is arranged in the support area and/or adjacent thereto. In this manner, an electric field can be created on the surface of the object carrier, which exerts a force on the biological cells, their dielectricity constants differing from those of the culture medium in which they are arranged, which leads the cells to the poration tool.
With a preferred embodiment of the invention, the object carrier is constructed approximately as a plate-shaped carrier element which has the support area on a flat side. The entry channel penetrates the object carrier proceeding from the support area to the reverse side of the object carrier facing away therefrom, and the object carrier has a wall weakening or thinning (attenuation) in the area of the entry channel, which in the direction of extension of the entry channel has a smaller dimension than a wall area adjacent to the wall attenuation. The entry channel is thus passed through the object carrier in the area of the wall attenuation and penetrates this, preferably at right angles to the plane stretching across the support area. In this manner, an especially short entry channel is obtained. The entry channel can, for example, be connected with a pump or a suction facility arranged on the reverse side of the object carrier. The wall area of the object carrier bordering on the wall attenuation, and preferably encircling it, has a greater wall thickness than the wall attenuation which improves the mechanical stability of the object carrier.
It is advantageous if the wall attenuation is arranged on a preferably funnel-shaped form formation in situated on the reverse side of the object carrier facing away from the support area. The support area can then be arranged in a plane extending up to and over the wall attenuation, so that the cells can better accumulate thereon.
An especially advantageous refinement of the invention provides that, in the support area, several poration tools having at least one entry channel are preferably arranged as arrays. With such a device, a plurality of cells closely adjacent to one another can be intracellularly manipulated, either at the same time or successively, whereby statistical fluctuations can be eliminated. Optionally, a multiplexer can be incorporated into the object carrier with which a plurality of electroporation electrodes can be controlled alteratively one after the other, whereby the number of supply leads to the object carrier is correspondingly reduced.